Biomaterial derived from vertebrate liver tissue

ABSTRACT

A tissue graft composition comprising liver basement membrane is described. The graft composition can be implanted to replace or induce the repair of damaged or diseased tissues.

[0001] This invention was made with U.S. Government support under Grant#HD-31425 awarded by the National Institute of Health. The U.S.Government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to a tissue graft composition andmethods for its preparation and use. More particularly, the presentinvention is directed to non-immunogenic tissue graft compositionscomprising the basement membrane of liver and the use of same to promoteendogenous tissue growth in vivo and to support the growth anddifferentiation of eukaryotic cells cultured in vitro.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] There has been much research effort directed to finding naturaland synthetic materials having the requisite properties for use astissue grafts. Surprisingly, it has been found that basement membranes(stroma) prepared from liver tissue of warm-blooded vertebrates byremoving cellular components of the liver tissue exhibit certainmechanical and biotropic properties similar to that which has beenreported for intestinal submucosal tissue in U.S. Pat. Nos. 4,902,508;5,281,422; and 5,275,826. It can be substituted for intestinalsubmucosa-tissue in most, if not all, of the applications previouslyreported for intestinal submucosa, including enhancing wound healing,promoting endogenous tissue growth, stimulating cell proliferation andinducing cell differentiation.

[0004] The basement membrane of the liver is an extracellular matrixdistinct from submucosal extracellular matrices. The liver basementmembrane does not support an overlaying mucosa and is devoid of thelaminate tissue structure in which submucosal extracellular matricesreside. The liver plays a central role in numerous regulatory processesin the body, including glucose metabolism, insulin regulation, anabolicprocesses for the musco-skeletal system and central nervous system, andthe maintenance of appropriate levels of circulating proteins essentialfor day to day homeostasis.

[0005] In one embodiment of the present invention, liver basementmembranes are used to manufacture a non-immunogenic tissue graftcomposition for use in the repair of damaged or diseased tissues. Thetissue graft composition of the present invention comprises the basementmembrane of organ tissue of a warm-blooded vertebrate, for example,liver tissue, substantially free, preferably devoid, of all cells (e.g.,hepatocytes and bile ductal cells) of said warm-blooded vertebrate. Thepresent tissue graft composition can be implanted, or fluidized andinjected, into a vertebrate host to contact damaged or defective tissuesand induce the repair or replacement of said tissues. The compositionsof the present invention can also be applied as a component of a wounddressing (ointment or bandage) in fluidized or solid form for topicalapplication to promote wound healing. Alternatively, the liver tissuederived extracellular matrix can be utilized as a cell growth substratefor growing eukaryotic cells in vitro.

DETAILED DESCRIPTION OF THE INVENTION

[0006] The tissue graft composition of the present invention comprisesliver basement membrane prepared by separating same from the nativelyassociated cellular components of liver tissue of a warm-bloodedvertebrate. The preparative techniques described below provide anextracellular matrix composition consisting essentially of liverbasement membrane substantially free of any cellular components. Thesecompositions are referred to herein generically as liver basementmembrane(s) (LBM). Other organ tissue sources of basement membrane foruse in accordance with this invention include spleen, lymph nodes,salivary glands, prostate, pancreas and other secreting glands.

[0007] Basement membrane for use in the graft composition of thisinvention is typically prepared from liver tissue harvested from animalsraised for meat production, including, for example, pigs, cattle andsheep or other warm-blooded vertebrates. Thus, there is an inexpensivecommercial source of liver tissue for use in preparation of the tissuegraft compositions in accordance with the present invention. Inaccordance with one embodiment, a composition comprising liver basementmembranes is prepared from liver tissue of a warm-blooded vertebrate.This composition is useful as a non-immunogenic tissue graft capable ofinducing endogenous tissue growth when implanted in warm-bloodedvertebrates. In one embodiment, the composition comprises anextracellular matrix consisting essentially of liver basement membranedevoid of endogenous cells associated with the source vertebrate livertissue used to prepared the composition.

[0008] The preparation of liver basement membrane from of liver tissueof a warm-blooded vertebrate in accordance with the present invention iscarried out by removing the cellular components from liver tissue.Ideally the process is carried out to separate the cells from thebasement membranes without damaging, or at least minimizing disruptionor damage to, the basement membrane tissue. Removal of the cellularcomponents from the liver extracellular matrix allows the preparation ofa graft composition that is non-immunogenic, and thus does not induce ahost immune response when the graft composition is implanted into ahost. In general, the method for preparing a tissue graft compositionfrom warm-blooded vertebrate liver tissue comprising the steps oftreating the liver tissue with a cell dissociation solution for a periodof time sufficient to release the cellular components of the livertissue from the extracellular components without substantial disruptionof the extracellular components, and separating the cellular componentsfrom said extracellular components. Typically the cell dissociationsolution comprises a chaotropic agent or an enzyme or both.

[0009] The first step in preparing LBM in accordance to one embodimentof the present invention comprises slicing a segment of liver tissueinto pieces (e.g., strips or sheets) to increase the surfacearea-to-volume ratio of the liver tissue. In one embodiment the livertissue is sliced into a series of sheets each having a thickness ofabout 50 to about 500 microns, more preferably about 250 to about 300microns. Freshly harvested liver tissue can be sliced using a standardmeat slicer, or the tissue can be frozen and sliced with acryomicrotone. The thin pieces of liver tissue are then treated with asolution that releases component liver cells from the associatedextracellular basement membrane matrix.

[0010] In accordance with one embodiment the liver tissue is treatedwith a solution comprising an enzyme, for example, a protease, such astrypsin or pepsin. Because of the collagenous structure of the liverbasement membrane and the desire to minimize degradation of the membranestructure during cell dissociation, collagen specific enzyme activityshould be minimized in the enzyme solutions used in thecell-dissociation step. In addition, the liver tissue is typically alsotreated with a calcium chelating agent or chaotropic agent such as amild detergent such as Triton 100. Thus, in one embodiment of thisinvention liver tissue is treated by suspending slices or strips of thetissue in a cell-dissociation solution containing enzyme(s) andchaotropic agent(s). However, the cell dissociation step can also beconducted using a calcium chelating agent or chaotropic agent in theabsence of an enzymatic treatment of the tissue.

[0011] In one preferred embodiment the cell-dissociation step is carriedout by suspending liver tissue slices in an agitated solution containingabout 0.05 to about 2%, more typically about 0.1 to about 1% by weightprotease, optionally containing a chaotropic agent or a calciumchelating agent in an amount effective to optimize release andseparation of cells from the basement membrane without substantialdegradation of the membrane matrix.

[0012] After contacting the liver tissue with the cell-dissociationsolution for a time sufficient to release all cells from the matrix, theresulting liver basement membrane is rinsed one or more times withsaline and optionally stored in a frozen hydrated state or a partiallydehydrated state until used as described below. The cell-dissociationstep may require several treatments with the cell-dissociation solutionto release substantially all cells from the basement membrane. In oneembodiment liver tissue is treated with a protease solution to removethe component cells, and the resulting extracellular matrix material(basement membrane) is further treated to remove or inhibit any residualenzyme activity. For example, the resulting basement membrane can beheated or treated with one or more protease inhibitors.

[0013] Liver basement membrane in accordance with this invention can befluidized (converted to an injectable or powder form) in a mannersimilar to the preparation of fluidized intestinal submucosa, asdescribed in U.S. Pat. No. 5,275,826 the disclosure of which isexpressly incorporated herein by reference. Liver basement membrane(devoid of cells from the source liver tissue) is comminuted by tearing,cutting, grinding, shearing and the like. Grinding the liver basementmembrane in a frozen or freeze-dried state is preferred although goodresults can be obtained as well by subjecting a suspension of liverbasement membrane to treatment in a high speed (high shear) blender anddewatering, if necessary, by centrifuging and decanting excess water.Additionally, the comminuted fluidized tissue can be solubilized byenzymatic digestion with a protease, for example a collagenase and orother appropriate enzyme, such as glycanase, or other enzyme thatdisrupts the matrix structural components, for a period of timesufficient to solubilize said tissue and form a substantiallyhomogeneous solution.

[0014] The present invention also contemplates the use of powder formsof liver basement membrane. In one embodiment a powder form of liverbasement membrane is prepared by pulverizing liver basement membranesubmucosa tissue under liquid nitrogen to produce particles ranging insize from 0.1 to 1 mm². The particulate composition is then lyophilizedovernight and sterilized to form a solid substantially anhydrousparticulate composite. Alternatively, a powder form of liver basementmembrane can be formed from fluidized liver basement membranes by dryingthe suspensions or solutions of comminuted/liver basement membrane. Thedehydrated forms have been rehydrated and used as cell culturesubstrates as described below without any apparent loss of their abilityto support cell growth.

[0015] To determine the components of the isolated liver basementmembranes of the present invention, the membranes have been extractedand the isolated fractions analyzed by Western blot analysis. LBM wasextracted with guanidine hydrochloride or urea, as described in Example4 and Western blot analysis, using antibodies directed against variousspecific growth factors, indicated the presence of basic growthfibroblast growth factor (bFGF), hepatocyte growth factor (HGF) andepidermal growth factor

[0016] The present liver basement membrane compositions may besterilized using conventional sterilization techniques including tanningwith glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the material is preferably used. For instance,it is believed that strong gamma radiation may cause loss of strength inthe graft material. Preferred sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation and gas plasmasterilization; peracetic acid sterilization being the most preferredmethod. In particular, LBM has been disinfected and sterilized throughthe use of either peracetic acid or one megarad of gamma irradiationwithout adversely effecting the mechanical properties or biologicalproperties of the tissue. The treatment with peracetic acid is conductedat a pH of about 2 to about 5 in an aqueous ethanolic solution (2-10%ethanol by volume) at a peracid concentration of about 0.03 to about0.5% by volume. Typically, after the graft composition has beensterilized, the composition is wrapped in a porous plastic wrap andsterilized again using electron beam or gamma irradiation sterilizationtechniques.

[0017] In accordance with one embodiment, liver basement membrane isused as, or used to prepare, tissue graft compositions of the presentinvention. Such tissue graft compositions lend themselves to a widevariety of surgical applications relating to the repair or replacementof damaged tissues, including, for example the repair of connectivetissues. Connective tissues for the purposes of the present inventionincludes bone, cartilage, muscle, tendons, ligaments, and fibrous tissueincluding the dermal layer of skin.

[0018] In accordance with this invention, the present tissue graftcompositions are used advantageously to induce the formation ofendogenous tissue at a desired site in a warm blooded vertebrate.Compositions comprising an extracellular matrix, consisting essentiallyof liver basement membrane, can be administered to a vertebrate host inan amount effective to induce endogenous tissue growth at a site in thehost in need of same due to the presence of damaged or diseased tissue.The present liver tissue derived tissue graft compositions can beadministered to the host in either solid form, by surgical implantation,or in fluidized form, by injection.

[0019] The liver basement membrane segments can be used in accordancewith this invention as a tissue graft construct for use in the repair orreplacement of connective tissues using the same procedures describedfor use of intestinal submucosa in U.S. Pat. Nos. 5,281,422 and5,352,463, each expressly incorporated herein by reference.

[0020] The tissue graft compositions formed and used in accordance withthis invention, upon implantation, undergo biological remodeling. Theyserve as a rapidly vascularized matrix for supporting the growth of newendogenous connective tissue. When used as a tissue graft material liverbasement membrane is expected to be trophic for host tissues with whichit is attached or otherwise associated in its implanted environment.

[0021] The liver basement membrane graft composition can be formed in avariety of shapes and configurations, for example, to serve as aligament or tendon replacement or a patch for a broken or severed tendonor ligament. Preferably, the segment is shaped and formed to have alayered or even a multilayered configuration with at least the oppositeend portions and/or opposite lateral portions being formed to havemultiple layers of the graft material to provide reinforcement forattachment to physiological structures, including bone, tendon,ligament, cartilage and muscle. In a ligament replacement application,opposite ends are attached using standard surgical technique to firstand second bones, respectively, the bones typically being articulated asin the case of a knee joint.

[0022] The end portions of the liver basement membrane graft compositioncan be formed, manipulated or shaped to be attached, for example, to abone structure in a manner that will reduce the possibility of grafttearing at the point of attachment. Preferably the material can befolded or to provide multiple layers for gripping, for example, withspiked washers or staples.

[0023] Alternatively, the liver basement membrane graft material may befolded back on itself to join the end portions to provide a firstconnective portion to be attached, for example, to a first bone and abend in the intermediate portion to provide a second connective portionto be attached to a second bone articulated with respect to the firstbone. For example, one of the end portions may be adapted to be pulledthrough a tunnel in, for example, the femur and attached thereto, whilethe other of the end portions may be adapted to be pulled through atunnel in the tibia and attached thereto to provide a substitute for thenatural cruciate ligament, the segment being adapted to be placed undertension between the tunnels to provide a ligament function, ie, atensioning and positioning function provided by a normal ligament.

[0024] The liver basement membranes of the present invention have beenimplanted in rabbits and in dogs to serve as Achilles tendon replacementgraft constructs. Two rabbits and two dogs were each implanted withAchilles tendon replacement LBM graft constructs using a similarprocedure as used for intestinal submucosal tissue as describe in U.S.Pat. No. 4,902,508. The experiments demonstrated that LBM graftconstructs could support the regeneration of the Achilles tendon.

[0025] During preparation of the liver basement membrane, the tissue iscut or sliced into pieces/slices. After the cell-dissociation processingstep the individual segments of liver basement membrane can beoverlapped with one another and bonded together using standardtechniques known to those skilled in the art, including the use ofsutures, crosslinking agents, and adhesives or pastes. Alternatively, inone embodiment, the overlapped layers of submucosal tissue are fused toone another by applying pressure to the overlapped regions underdehydrating conditions. The term “dehydrating conditions” is defined toinclude any mechanical or environmental condition which promotes orinduces the removal of water from the submucosal tissue. To promotedehydration of the compressed submucosal tissue, at least one of the twosurfaces compressing the tissue is water permeable. Dehydration of thetissue can optionally be further enhanced by applying blotting material,heating the tissue or blowing air across the exterior of the compressingsurfaces. Accordingly, multilayer liver basement membrane constructs canbe prepared to provide tissue graft compositions of enhanced strength.

[0026] In addition, by overlapping a portion of one piece of liverbasement membrane with a portion of at least one additional piece ofliver basement membrane and bonding the overlapped layers to oneanother, large area sheets of liver basement membrane can be formed. Inone embodiment, during formation of the large area sheets of tissue,pressure is applied to the overlapped portions under dehydratingconditions by compressing the overlapped tissue segments between twosurfaces. The two surfaces can be formed from a variety of materials andin any shape depending on the desired form and specification of thetargeted graft construct. Typically the two surfaces are formed as flatplates but they can also include other shapes such as screens, opposedcylinders or rollers and complementary nonplanar surfaces. Each of thesesurfaces can optionally be heated or perforated. In preferredembodiments at least one of the two surfaces is water permeable. Theterm water permeable surface as used herein includes surfaces that arewater absorbent, microporous or macroporous. Macroporous materialsinclude perforated plates or meshes made of plastic, metal, ceramics orwood.

[0027] The liver basement membrane can be compressed in accordance withone embodiment by placing the overlapped portions of the strips ofcell-dissociated liver membrane on a first surface and placing a secondsurface on top of the exposed membrane surface. A force is then appliedto bias the two surfaces towards one another, compressing the membranecomposition between the two surfaces. The biasing force can be generatedby any number of methods known to those skilled in the art including thepassage of the apparatus through a pair of pinch rollers (the distancebetween the surface of the two rollers being less than the originaldistance between the two plates), the application of a weight on the topplate, and the use of a hydraulic press or the application ofatmospheric pressure on the two surfaces.

[0028] In one preferred embodiment, a multi-layered liver basementmembrane graft material is prepared without the use of adhesives orchemical pretreatments by compressing at least the overlapped portionsof submucosal tissue under conditions that allow dehydration of thematerial concurrent with the compression of the tissue. To promotedehydration of the compressed material, at least one of the two surfacescompressing the tissue is water permeable. Dehydration can optionally befurther enhanced by applying blotting material, heating the material orblowing air across the exterior of the two compressing surfaces. Thecompressed multi-layered liver basement membrane material can be removedfrom the two surfaces as a unitary compliant large area graft construct.The construct can be further manipulated (i.e., cut, folded, sutured,etc.) to suit various medical applications where the liver basementmembrane material is required.

[0029] A vacuum can optionally be applied to liver basement membraneduring the compression procedure. The applied vacuum enhances thedehydration of the tissue and may assist the compression of the tissue.Alternatively the application of a vacuum may provide the solecompressing force for compressing the overlapped portions of themultiple layers of liver basement membranes. For example, in oneembodiment the overlapped liver basement membrane is laid out betweentwo surfaces, preferably one of which is water permeable. The apparatusis covered with blotting material, to soak up water, and a breatherblanket to allow air flow. The apparatus is then placed in a vacuumchamber and a vacuum is applied, generally ranging from 35.6-177.8 cm ofHg (0.49-2.46 Kg/cm²) and more preferably the vacuum applied isapproximately 129.5 cm of Hg (1.76 Kg/cm²). Optionally a heating blanketcan be placed on top of the chamber to heat the liver basement membraneduring compression. Chambers suitable for use in this embodiment areknown to those skilled in the art and include any device that isequipped with a vacuum port. The resulting drop in atmospheric pressurecoacts with the two surfaces to compress the tissue and simultaneouslydehydrate the compressed tissue.

[0030] In an alternative embodiment of the present invention, liverbasement membrane can be utilized in a method and composition forsupporting the proliferation and induction of tissue differentiation ofeukaryotic cells cultured in vitro. Generally the method comprises thestep of contacting eukaryotic cells, in vitro, with a liver basementmembrane composition under conditions conducive to eukaryotic cellgrowth. The term “contacting” as used herein with reference to cellculture is intended to include both direct and indirect contact, forexample in fluid communication, of the liver basement membranecomposition and the cultured cells. The term “conditions conducive toeukaryotic cell growth” as used herein refers to the environmentalconditions, such as sterile technique, temperature and nutrient supply,that are considered optimal for eukaryotic cell growth under currentlyavailable cell culture procedures. Although optimum cell cultureconditions used for culturing eukaryotic cells depend somewhat on theparticular cell type, cell growth conditions are generally well known inthe art. However a number of differentiated cell types are stillconsidered difficult to culture (i.e., islets of Langerhans,hepatocytes, chondrocytes, osteoblasts, etc.).

[0031] Applicants have discovered that compositions comprising liverbasement membrane prepared in accordance with this invention can be usedfor supporting growth or proliferation of eukaryotic cells in vitro. Inaccordance with one embodiment a liver tissue derived composition forsupporting the growth of a cell population is prepared from liver tissueof a warm-blooded vertebrate. The composition comprises isolated liverbasement membrane devoid of source liver tissue endogenous cells andadded nutrients to support the growth of said cell population in vitro.In addition fluidized forms of liver basement membrane can be used tocoat culture-ware with a matrix comprising liver basement membranedevoid of source liver tissue endogenous cells. Thus liver basementmembrane can be used as a cell growth substrate in a variety of forms,including a sheet-like configuration, as a gel matrix, as an additivefor art-recognized cell/tissue culture media, or as coating forculture-ware to provide a more physiologically relevant substrate thatsupports and enhances the proliferation of cells.

[0032] The liver basement membrane, due to its honeycomb-like structure(that which remains after cell-dissociation) provides a high surfacearea for cell adhesion and also induces cell differentiation. Scanningelectron images indicate that the isolated liver basement membrane isvery porous. When fetal rat cells are cultured on liver basementmembranes that are retained in their nature three dimensional shape,scanning electron images reveal that the fetal rat cells form confluentsheets on the liver cell substrate and also invade into the LBM matrix.The membrane material is preferably sterilized prior to use in cellculture applications, however nonsterile material can be used ifantibiotics are included in the cell culture system.

[0033] In one preferred embodiment cells are seeded directly onto sheetsof liver basement membrane under conditions conducive to eukaryotic cellproliferation. The highly porous nature of the liver basement membraneallow diffusion of cell nutrients throughout the membrane matrix. Thus,cells can be cultured on or within the liver basement membrane matrix.

[0034] In another embodiment of the present invention, cell growthsubstrates are formed from fluidized forms of liver basement membrane.The fluidized tissue can be gelled to form a solid or semi-solid matrix.The viscosity of fluidized tissue for use in accordance with thisinvention can be manipulated by controlling the concentration of thetissue component and the degree of hydration. The viscosity can beadjusted to a range of about 2 to about 300,000 cps at 25° C. Higherviscosity formulations, for example, gels, can be prepared from thedigest solutions by adjusting the pH of such solutions to about 6.0 toabout 7.4. Eukaryotic or prokaryotic cells can then be seeded directlyon the surface of the matrix and cultured under conditions conducive toeukaryotic cell proliferation.

[0035] The cell growth substrates of the present invention can becombined with nutrients; including minerals, amino acids, sugars,peptides, proteins, or glycoproteins that facilitate cellularproliferation, such as laminin and fibronectin and growth factors suchas epidermal growth factor, platelet-derived growth factor, transforminggrowth factor beta, or fibroblast growth factor. In one embodimentfluidized or powder forms of liver basement membrane can be used tosupplement standard eukaryotic culture media to enhance the standardmedia's capacity for sustaining and inducing the proliferation of cellscultured in vitro.

[0036] In accordance with the present invention there is provided a cellculture composition for supporting growth in vitro of an eukaryotic cellpopulation in combination with liver basement membrane of a warm-bloodedvertebrate. The composition comprises liver basement membranesubstantially free of the original associated endogenous cells. Thecomposition can further comprise nutrients, and growth factors requiredfor optimal growth of the cultured cells. The liver basement membranecell culture substrate can be used with commercially available cellculture liquid media (both serum based and serum free). Proliferatingcells can either be in direct contact with the liver basement membraneor they can simply be in fluid communication with the liver basementmembrane.

[0037] It is anticipated that cell growth compositions utilizing theliver basement membrane composition of the present invention can be usedto stimulate proliferation of undifferentiated stems cells as well asdifferentiated cells such as islets of Langerhans, hepatocytes andchondrocytes. Furthermore, the described cell growth composition isbelieved to support the growth of differentiated cells while maintainingthe differentiated state of such cells. Several primary cell lines havebeen grown on LBM derived cell culture substrates in vitro, includingprimary cell lines derived from the cruciate ligament. Primary celllines derived from the cruciate ligament show an approximate doubling ofthe growth rate compared to when these cells are grown on plastic.

[0038] It is anticipated that liver basement membrane is capable ofinducing host tissue proliferation, remodeling and regeneration ofappropriate tissue structures upon implantation in a number ofmicroenvironments in vivo (e.g., tendon, ligament; bone, articularcartilage, artery, and vein). In one embodiment of the present inventionthe tissue replacement capabilities of graft compositions comprisingliver basement membrane of warm-blooded vertebrates are further enhancedor expanded by seeding the tissue with various cell types, prior toimplantation. For example, a liver basement membrane derived cellculture substrate may be seeded with endothelial cells or keratinocytesfor use as a vascular graft or skin replacement, respectively.Alternatively, the liver basement membrane can be seeded withmesenchymal cells (stem cells) initially for expansion of the cellpopulation and thereafter for implantation into a host. Liver basementmembrane can also serve as a delivery vehicle, either in fluidize formor in its native solid form, for introducing various cell populations,including genetically modified cells, to a specific location in a host.Optionally, after the liver basement membrane have been seeded witheukaryotic cells, the graft composition can be subjected to conditionsconducive to the proliferation of eukaryotic cells to further expand thepopulation of the seeded cells prior to implantation of the graft intothe host.

[0039] In another embodiment, compositions comprising liver basementmembrane and a proliferating cell population can be encapsulated in abiocompatible matrix for implantation into a host. The encapsulatingmatrix can be configured to allow the diffusion of nutrients to theencapsulated cells while allowing the products of the encapsulated cellsto diffuse from the encapsulated cells to the host cells. Suitablebiocompatible polymers for encapsulating living cells are known to thoseskilled in the art. For example a polylysine/alginate encapsulationprocess has been previously described by F. Lim and A. Sun (Science Vol.210 pp. 908-910). Indeed, the present liver basement membranecomposition itself could be used advantageously to encapsulate aproliferating cell population in accordance with this invention forimplantation as an artificial organ.

EXAMPLE 1 Preparation of Liver Basement Membrane

[0040] 2 mM EDTA Chaotropic Solution Used In The Experiment 140 mM NaCl5 mM KCl 0.8 mM MgSO₄ 0.4 mM KH₂HPO₄ 2 mM EDTA 25 mM NaHCO₃

[0041] Procedure:

[0042] Preparation of liver slices:

[0043] Liver frozen in −70° C. was sliced with a cryomicrotone to athickness of about 50 μM. The slices of liver tissue were then subjectedto enzymatic treatment (trypsin) with a chaotropic solution (samples 1and 2), with enzyme alone (samples 3 and 4), or with a chaotropicsolution alone (sample 5), as indicated below. Sample # Treatment 1)0.05% Trypsin in 2 mM EDTA solution 2) 0.1% Trypsin in 2 mM EDTAsolution 3) 0.05% Trypsin in 2 mM PBS 4) 0.1% Trypsin in 2 mM PBS 5) 2mM EDTA solution

[0044] Liver slices were placed in five 50 ml tubes, each of whichcontained 25 mL of a different buffered enzyme treatment solution. Theliver tissue was incubated at 37° C. in water bath with gentle shakingfor 1 hour. The liver slices were washed twice with PBS withagitation/shaking for 1 hour at room temperature. The above enzymatictreatment steps were repeated three times.

[0045] The wash buffers were collected and spin them down in 2000 rpmfor 10 min. The pellet was suspended and an equal amount of trypan bluewas added to identify any remaining cells. The material was checked forpresence of cells under microscope.

EXAMPLE 2 Mechanical Properties of Isolated Liver Basement Membrane

[0046] Porosity of a graft material is typically measured in terms of mlof water passed per cm²min⁻¹ at a pressure of 120 mm Hg. The average“porosity index” established for two separate specimens of LBM was 1162.The suture retention strength of LBM is approximately 68 grams. Thematerial appears to be anisotropic, with the suture strength beingapproximately the same in all directions.

EXAMPLE 3 Growth and Differentiation of Various Cell Lines on LiverBasement Membrane

[0047] In the present study, the growth and differentiation of threedifferent cell lines on liver basement membrane (LBM) was investigated.These tested cell lines included Swiss 3T3, a fibroblast cell line, ROS.17/2.8, an osteosarcoma cell line and PC12, a neuronal cell line. Allcells were seeded on a collagen coated plate. The seeding densities forSwiss 3T3 and ROS cells was 20×10⁴ cells/ml, whereas the PC12 was seededat 5×10⁴ cells/ml. In order to compare the growth and differentiation ofthese cells lines on LBM and small intestinal submucosa (SIS), the samecells with same densities were also seeded on SIS. In addition, apositive control for PC12 cells was achieved by adding nerve growthfactor (NGF) 50 ng/ml to the cells. All experiments were carried out induplicate. After five days one sample from each cell line seeded on SISand LBM was prepared for histology.

[0048] Preliminary observations indicate growth of all three cell linesand differentiation of ROS and PC12 cell lines on both substrates. Alight microscopic observation of PC12 cells indicated a greater degreeof differentiation on LBM compared to the cells grown on SIS. Inaddition, ROS and 3T3 cells appeared to grow/differentiate as well asthey do on SIS. Due to the transparent nature of LBM, it was difficultto quantify the growth and differentiation of the cells, but thehistological examination will allow a more detailed assessment.

EXAMPLE 4 Preparation of Extracts of LBM

[0049] Extraction buffers used for these studies included 4M guanidineand 2M urea each prepared in 50 mM Tris-HCl, pH 7.4. The powder form ofLBM was suspended in the relevant extraction buffer (25% w/v) containingphenylmethyl sulphonyl fluoride, N-ethylmaleimide, and benzamidine(protease inhibitors) each at 1 mM and vigorously stirred for 24 hoursat 4° C. The extraction mixture was then centrifuged at 12,000×g for 30minutes at 4° C. and the supernatant collected. The insoluble materialwas washed briefly in the extraction buffer, centrifuged, and the washcombined with the original supernatant. The supernatant was dialyzedextensively in Spectrapor tubing (MWCO 3500, Spectrum MedicalIndustries, Los Angeles, Calif.) against 30 volumes of deionized water(9 changes over 72 hours). The dialysate was centrifuged at 12,000×g toremove any insoluble material and the supernatant was used immediatelyor lyophilized for long term storage.

[0050] Western blot analysis with antibodies specific for bFGF HGF andEGF detected a corresponding reactive band for each of the antibodies,confirming the presence of these growth factors in LBM.

1. A composition comprising liver basement membrane of a warm-bloodedvertebrate, wherein the liver basement membrane is substantially free ofcells of said warm-blooded vertebrate.
 2. The composition of claim 1wherein the liver basement membrane is fluidized.
 3. The composition ofclaim 1, wherein the liver basement membrane is dried and in powderform.
 4. A composition prepared from liver tissue of a warm-bloodedvertebrate, wherein said composition is useful as a non-immunogenictissue graft capable of inducing endogenous tissue growth when implantedin warm-blooded vertebrates, said composition comprising anextracellular matrix consisting essentially of liver basement membranedevoid of endogenous cells associated with said liver tissue.
 5. The useof liver basement membrane to manufacture a non-immunogenic tissue graftcomposition for use in the repair of damaged or diseased tissues.
 6. Amethod for inducing the formation of endogendus tissue at a site in needof endogenous tissue growth in a warm blooded vertebrate, said methodcomprising implanting a graft composition comprising an extracellularmatrix consisting essentially of basement membrane of liver tissue of awarm-blooded vertebrate in an amount effective to induce endogenoustissue growth at the site the graft composition is administered.
 7. Themethod of claim 6, wherein the graft composition is fluidized and isadministered by injection into the warm-blooded vertebrate.
 8. Themethod of claim 6, wherein the graft composition is administered bysurgically implanting the composition into the warm-blooded vertebrate.9. A method for preparing a tissue graft composition from warm-bloodedvertebrate liver tissue having both cellular and extracellularcomponents, said method comprising the steps of treating the livertissue with a cell dissociation solution for a period of time sufficientto release the cellular components of the liver tissue from theextracellular components without substantial disruption of theextracellular components, and separating the cellular components fromsaid extracellular components.
 10. The method of claim 9 wherein thecell dissociation solution comprises a chaotropic agent.
 11. The methodof claim 9 wherein the cell dissociation solution comprises a protease.12. The method of claim 9 wherein the cell dissociation solutioncomprises EDTA and trypsin.
 13. The method of claim 9 wherein the livertissue is sliced into sheets or strips of liver tissue before the livertissue is treated with the dissociation solution.
 14. The method ofclaim 13 where the liver tissue is sliced into sheets or strips having athickness of up to abut 500μ.
 15. A liver tissue derived composition forsupporting the growth of a cell population, said composition comprisingisolated liver basement membrane devoid of source liver tissueendogenous cells; and added nutrients to support the growth of said cellpopulation in vitro.
 16. A liver tissue derived composition forsupporting the growth of a cell population, said composition comprisingculture-ware coated with a matrix comprising liver basement membranedevoid of source liver tissue endogenous cells.